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Canto flow cytometer

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The Canto flow cytometer is a laboratory instrument designed for the analysis of cells and particles in liquid samples. It utilizes the principles of flow cytometry to rapidly detect, identify, and count specific cell populations or particles based on their physical and fluorescent properties.

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Lab products found in correlation

Propidium iodide, by Merck Group (4 mentions) Fortessa flow cytometer, by BD (3 mentions) Facsdiva software, by BD (3 mentions) Annexin 5 binding buffer, by BD (2 mentions) Propidium iodide, by Thermo Fisher Scientific (2 mentions) Diva software, by BD (2 mentions) Trucount tube, by BD (2 mentions) Mitosox red, by Thermo Fisher Scientific (2 mentions) Software, by FlowJo (2 mentions) Anti ccr7 fitc 3d12, by BD (2 mentions) Anti cd45ra pe hi100, by BD (2 mentions) Anti cd4 pecy7 okt4, by BioLegend (2 mentions) Exoquick exosome isolation kit, by System Biosciences (2 mentions) Annexin 5 fitc, by Thermo Fisher Scientific (1 mentions) Annexin 5, by BD (1 mentions) Mouse igg1κ, by BD (1 mentions) T2200, by Merck Group (1 mentions) Anti annexin 5 antibody, by BioLegend (1 mentions) Brdu kit, by BD (1 mentions) 7 aad, by BD (1 mentions)

80 protocols using canto flow cytometer

1

Measuring Apoptosis in Multiple Myeloma Cells

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Apoptosis of MM cell lines was examined using the Annexin/PI assay as described before[25 (link), 31 (link), 36 (link), 37 (link)]. Briefly, cells were washed twice with annexin binding buffer (ABB) (10mM HEPES pH 7.4, 140mM NaCl, 2.5mM CaCl2). 100μl cells were treated with 3μl of annexin V-FITC (Caltag, Burlingame, CA, USA) for 15mins at room temperature. Cells were washed again with ABB and resuspended in 500μl of ABB containing 5μl of 1mg/ml propidium iodide (Sigma). Samples were then run on a Canto flow cytometer (BD Biosciences, San Jose, CA, USA).
For patient cells, apoptosis was examined using the Apo2.7 assay as described earlier[31 (link), 37 (link)]. Briefly, fresh bone marrow cells were ACK lysed, washed and resuspended in culture media and incubated with the drugs for the indicated time points. The cultures were harvested, washed once in PBS and resuspended in 1ml PBS/3%BSA. 100μl of cells were then stained with Apo 2.7 PE (Beckman Coulter, Miami, FL, USA) or isotype control for 15minutes. Cells were washed again in PBS and resuspended in 1% paraformaldehyde and stored in the dark at 4°C until run on the Canto flow cytometer (BD Biosciences)
Ramakrishnan V., Kimlinger T., Timm M., Haug J., Rajkumar S.V, & Kumar S. (2014). Multiple mechanisms contribute to the synergistic anti-myeloma activity of the pan-histone deacetylase inhibitor LBH589 and the rapalog RAD001. Leukemia research, 38(11), 1358-1366.
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2

Apoptosis Assessment of Myeloma Cells

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BDA-366 treated RPMI8226 or U266 cells (10,000 cells/well) were harvested after 48-hour culture. Apoptotic cell fractions were assessed with Annexin-V FITC and propidium iodide staining kit following the manufacturer's instruction (Invitrogen, USA). Alternatively, BDA-366 treated BM cells from MM patients were stained with anti-human CD38 (PE), CD45 (APC-Cy7), and CD138 (APC) antibodies (1:100 dilution) (BD Bioscience, San Jose, CA), and Annexin V (FITC). Primary myeloma cells were gated on CD45−CD38+CD138+ cells; and apoptotic cells were gated on Annexin V as previously described [41 (link)]. In addition, RPMI8226, U266 or primary MM cells treated with BDA-366 for 12 hours were harvested and intracellularly stained with anti- BCL2 BH3 domain-specific antibody (Abgent, San Diego, CA) [31 (link)]. All stained cells were subjected to fluorescence-activated cell sorting (FACS) analyses on a BD Canto Flow Cytometer. All FACS data were analyzed with FlowJo 9.1 software.
Deng J., Park D., Wang M., Nooka A., Deng Q., Matulis S., Kaufman J., Lonial S., Boise L.H., Galipeau J, & Deng X. (2016). BCL2-BH4 antagonist BDA-366 suppresses human myeloma growth. Oncotarget, 7(19), 27753-27763.
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3

Immunophenotyping of Neural Progenitors

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Cells were singularized with Accutase and fixed using cold 90% methanol. Primary antibodies against nestin (Santa Cruz Biotech SC23927), β-3 tubulin (Sigma T2200), and an isotype control (Mouse IgG1, κ; BD 550878) were used at a 1:500 dilution in 0.3% BSA and incubated overnight at 4°C. Secondary antibodies (AlexaFluor 488 and 568-conjugated) were used at 1:500 in 0.3% BSA and incubated for 1 hr at room temperature. Samples were analyzed on a BD Canto flow cytometer using BD FACSDiva software.
Laperle A., Masters K.S, & Palecek S.P. (2014). Influence of substrate composition on human embryonic stem cell differentiation and extracellular matrix production in embryoid bodies. Biotechnology progress, 31(1), 212-219.
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4

Cell Cycle and Apoptosis Analysis

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Cell cycle analysis was performed using a BrdU kit (BD Biosciences, 552598). Cells were exposed to BrdU for 30 min and BrdU incorporation was assessed according to manufacturer’s instructions. For analysis of apoptosis, cells were incubated with anti-Annexin V antibody (Biolegend, 556420, 1:100) in Annexin V binding buffer (BD Biosciences) for 15 min. Cells were then stained with 7-AAD (BD Biosciences). Both BrdU and Annexin V staining data were acquired using a BD Canto flow cytometer.
Cell cycle analysis on HSPCs from BM was performed with freshly isolated HSPCs. Lin-depleted BM cells were stained for HSPC markers, then fixed and permeabilized followed by staining with anti-Ki-67-APC antibodies (eBio 50–5698–80). Cell cycle analysis is performed along with DAPI staining for DNA content and assessed on a BD Fortessa flow cytometer.
Balcerek J., Jiang J., Li Y., Jiang Q., Holdreith N., Singh B., Chandra V., Lv K., Ren J.G., Rozenova K., Li W., Greenberg R.A, & Tong W. (2018). Lnk/Sh2b3 deficiency restores hematopoietic stem cell function and genome integrity in Fancd2 deficient Fanconi anemia. Nature Communications, 9, 3915.
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5

Immunophenotyping and Cell Cycle Analysis of Cultured hPSCs

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hPSCs cultured on the aforementioned coatings were singularized with Accutase and fixed in cold 90% methanol. Primary antibodies against annexin V (1:500, Santa Cruz Biotechnology, catalog no. SC74458), Nanog (1:500, Cell Signaling Technology, catalog no. 4893), cleaved caspase-3 (1:250, Cell Signaling Technology, catalog no. 9664), Ki67 (1:500, Abcam, 15580), and an isotype control (1:500 and 1:250, mouse immunoglobulin G1 [IgG1], BD Biosciences, catalog no. 550878) were prepared in 0.3% BSA in PBS and incubated with samples overnight at 4°C. After washing, samples were incubated for 1 hr at room temperature with Alexa Fluor 488- or 568-conjugated goat anti-mouse or goat anti-rabbit secondary antibodies (Life Technologies, catalog nos. A10667 and A11031, respectively) diluted 1:500 in 0.3% BSA. Samples were analyzed on a BDCanto flow cytometer using BD FACSdiva software, and negative control gating was performed on samples treated with an isotype control primary antibody and a secondary antibody identical to that used for samples. For cell-cycle analysis, samples were stained with propidium iodide (Life Technologies), and cell-cycle distribution was determined using the ModFit LT version 4 software package from Verity Software House.
Laperle A., Hsiao C., Lampe M., Mortier J., Saha K., Palecek S.P, & Masters K.S. (2015). α-5 Laminin Synthesized by Human Pluripotent Stem Cells Promotes Self-Renewal. Stem Cell Reports, 5(2), 195-206.
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6

Immunophenotyping of Classic AT Patients

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Peripheral blood samples and clinical data were collected from 5 classic AT patients, who are under follow up at the Radboud University Medical Center (Table S1). Ethical approval was obtained from the Ethical Committee Arnhem/Nijmegen with protocol registration number 2011/304. Written informed consent was obtained from all patients. Five healthy control subjects were also included in our study (B17.001). Leukocyte subsets (absolute leukocyte counts and differential) were measured by using a Sysmex hematology analyzer and Trucount tubes (Becton Dickinson (BD), Franklin Lakes, NJ, USA) with a mixture of antibodies specific for lymphocyte subsets in freshly collected blood samples. Antibodies included CD45 (2D1, BD), CD3 (SK7; BD), CD19 (J4-119, Beckman Coulter, Brea, CA, USA), CD16 (3G8, BD) and CD56(C5.9, DAKO, Glostrup, Denmark). Samples were measured on a BD CANTO flow cytometer and analyzed with BD DIVA software. Peripheral blood mononuclear cells (PBMCs) were isolated using Ficoll density gradient centrifugation and cryo-stored before use.
Weitering T.J., Melsen J.E., van Ostaijen-ten Dam M.M., Weemaes C.M., Schilham M.W, & van der Burg M. (2021). Normal Numbers of Stem Cell Memory T Cells Despite Strongly Reduced Naive T Cells Support Intact Memory T Cell Compartment in Ataxia Telangiectasia. Frontiers in Immunology, 12, 686333.
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7

Naive CD8 T cell proliferation assay

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Naive CD8 T cells were isolated from the lymph nodes of approximately 12 weeks old OT-1 mice using a MACS CD8 II isolation kit. Anti-CD44-biotin antibody was added to biotinylated purification cocktail to remove memory T cells. Purified naive CD8 T cells were labelled with 5μM CTV and cultured at 10,000 cells /well in the presence of 1ng/mL IL-7 and stimulated with 10ng/mL SIINFEKL peptide and 100U/mL IL-2. Cells were incubated in a humidified atmosphere at 37°C and 5% CO2. At the time points indicated 10,000 beads and 0.2% propidium iodide were added to each well immediately prior to analysis on a BD Canto flowcytometer. Calculation of total cell numbers and cell numbers per division are described in [4 (link)].
Datasets b–cd40, b–cpg, and b–lps were obtained from the authors of [24 (link)]. These data are shown, respectively, in Supplementary Figures S4, S2 and S3 in reference [24 (link)]. Datasets t–il2, t–vv–qsc, and t–vv–tot were obtained from the authors of [4 (link)]. These data are shown, respectively, in Figures 2C, 1D and 4D in reference [4 (link)].
Kan A., Pavlyshyn D., Markham J.F., Dowling M.R., Heinzel S., Zhou J.H., Marchingo J.M, & Hodgkin P.D. (2016). Stochastic Measurement Models for Quantifying Lymphocyte Responses Using Flow Cytometry. PLoS ONE, 11(1), e0146227.
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8

Flow Cytometric Analysis of CB1 Receptor

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Validation of cell-surface expression of the HA-tagged wild-type and mutant CB1 receptors expressed in CHO-K1 cells was obtained by anti-HA antibody staining followed by quantitative flow cytometry and visually confirmed by confocal imaging. In brief, cells were serum-starved 30 min at 37 °C and collected in 5 mM EDTA and fixed with 4% paraformaldehyde for 10 min at 4 °C. Cells were washed twice with PBS and resuspended in PBS containing 1% FBS and 5 mM EDTA. Cells were incubated with anti-HA AlexaFluor488-conjugated antibody (1:1,000) for 30 min at 4 °C, washed twice with PBS and again resuspended in PBS containing 1% FBS and 5 mM EDTA. Fluorescence was recorded using a BD Canto flow cytometer (excitation/emission: 488/525 nm). Approximately 50,000 events were recorded for each cell line. Data are expressed as the percentage of positive-fluorescent cells from 50,000 events recorded (3×HA–CB1 CHO wild-type = 65%) and relative to 3×HA-CB1 wild-type CHO (100%). Untransfected CHO cells had 0% fluorescence. See Extended Data Fig. 4a for primers used to make mutant CB1 receptors and surface expression comparisons of all mutants reported herein. See Supplementary Fig. 1 for flow cytometry graphs.
Hua T., Vemuri K., Nikas S.P., Laprairie R.B., Wu Y., Qu L., Pu M., Korde A., Jiang S., Ho J.H., Han G.W., Ding K., Li X., Liu H., Hanson M.A., Zhao S., Bohn L.M., Makriyannis A., Stevens R.C, & Liu Z.J. (2017). Crystal structures of agonist-bound human cannabinoid receptor CB1. Nature, 547(7664), 468-471.
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9

Flow Cytometric Analysis of Tregs and Th17

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The frequency of Tregs was determined by staining fresh PBMC samples with CD4-FITC and CD25-allophycocyanin, and intracellularly staining for FoxP3-PE using rhesus macaque-specific antibodies (eBioscience, San Diego, CA, USA) according to the manufacturer’s instructions. Th17 frequencies were determined by surface staining previously frozen PBMCs with CD4-FITC and CD196-PE-Cy7, and intracellular staining with IL-17a-PE after 5-h stimulation with PMA/ionomycin + GolgiStop. Samples were run on a BD Canto flow cytometer, and data were analyzed using FlowJo software (Tree Star, Ashland, OR, USA).
Samelson-Jones B.J., Finn J.D., Favaro P., Wright J.F, & Arruda V.R. (2020). Timing of Intensive Immunosuppression Impacts Risk of Transgene Antibodies after AAV Gene Therapy in Nonhuman Primates. Molecular Therapy. Methods & Clinical Development, 17, 1129-1138.
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10

Quantifying B-cell Subsets in GBS

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Peripheral blood mononuclear cells (PBMC) were isolated from 67 GBS patients and 14 healthy controls (HC) using CPT tubes and directly stained with CD19‐PE/Cy7, CD27‐APC, CD38‐PerCP, IgD‐PE, and IgM‐FITC to determine the relative distribution of naïve, memory, and natural effector B cells, as well as plasmablasts within the total B‐cell population. Cells were measured on a BD Canto flow cytometer.
Simultaneously, a 100‐μL whole‐blood sample from EDTA blood (GBS patients: n = 45, HC: n = 14) was stained to quantify the total number of B cells per mL peripheral blood using TruCount tubes (BD Biosciences). Cells were stained for 20 min on ice using CD16‐FITC, CD56‐PE, CD19‐PE/Cy7, CD45‐BV510, and CD3‐AF700. Next, erythrocytes were lysed using 1 mL 0.155 mol/L NH4Cl, 10 mmol/L KHCO3, and 0.1 mmol/L Na2 EDTA.2H2O for 15 min on ice and the remaining cells were measured on a BD‐LSR II flow cytometer.
The total number of B cells was calculated using the ratio between CD45+ CD19+ cells and the number of beads measured in the same tube. Next, the absolute numbers of B‐cell subsets were calculated using the relative frequencies within the total CD19+ B‐cell population as described above.
Brem M.D., Jacobs B.C., van Rijs W., Fokkink W.J., Tio‐Gillen A.P., Walgaard C., van Doorn P.A., IJspeert H., van der Burg M, & Huizinga R. (2018). IVIg‐induced plasmablasts in patients with Guillain‐Barré syndrome. Annals of Clinical and Translational Neurology, 6(1), 129-143.
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